Active participation of Hsp90 in the biogenesis of the trimeric reovirus cell attachment protein σ1

The reovirus cell attachment protein, sigma 1, is a lollipop-shaped homotrimer with an N-terminal fibrous tail and a C-terminal globular head. Biogenesis of this protein involves two trimerization events: N-terminal trimerization, which occurs cotranslationally and is Hsp70/ATP-independent, and C-terminal trimerization, which occurs posttranslationally and is Hsp70/ATP-dependent. To determine if Hsp90 also plays a role in sigma 1 biogenesis, we analyzed sigma 1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature sigma 1 trimers (hydra-like intermediates with assembled N-termini and unassembled C termini) but not with mature trimers, The use of truncated sigma 1 further demonstrated that only the C-terminal half of sigma 1 associated with Hsp90. In the presence of the Hsp90 binding drug geldanamycin, N-terminal trimerization proceeded normally, but C-terminal trimerization was blocked. Geldanamycin did not inhibit the association of Hsp90 with sigma 1 but prevented the subsequent release of Hsp90 from the immature al complex. We also examined the status of p23, an Hsp90-associated co-chaperone. Like Hsp90, p23 only associated with immature sigma 1 trimers, and this association was mapped to the C-terminal half of sigma 1. However, unlike Hsp90, p23 was released from the sigma 1 complex upon the addition of geldanamycin. These results highlight an all-or-none concept of chaperone involvement in different oligomerization domains within a single protein and suggest a possible common usage of chaperones in the regulation of general protein folding and of steroid receptor activation.